Although the conditions for inducing virulence protein expression in vitro are different, both classical and El Tor biotypes of Vibrio cholerae have been reported to regulate the expression of virulence proteins such as cholera toxin (CT) and toxin-coregulated pili (Tcp) through the ToxR/S/T system. The transcription activator ToxR responds to environmental stimuli such as pH and temperature and activates the second transcriptional regulator ToxT, which upregulates expression of virulence proteins. In addition to the ToxR/S/T signalling system, V. cholerae has been proposed to utilize another two-component system VarS/VarA to modulate expression of virulence genes. Previous study has shown that VarA of the VarS/VarA system is involved in the regulation of virulence proteins in the classical V. cholerae O395 strain; however, no further analysis was performed concerning VarS. In this study, we constructed varS mutants derived from the classical O395 and El Tor C6706 strains and demonstrated that VarS is also involved in the expression of the virulence proteins CT and Tcp from the V. cholerae classical and El Tor strains. This expression is through regulation of ToxT expression in response to environmental changes due to different toxin-inducing conditions.